Various receptors on insect cells for Bacillus thuringiensis (B.t.) insecticidal toxin proteins are known in the art. See, e.g., U.S. Pat. Nos. 6,586,197; 6,429,360; 6,137,033; and 5,688,691. However, no known prior art taught or suggested administering fragments of cadherin-like proteins, especially fragments of B.t. toxin receptors, to insects.
Bacillus thuringiensis as an Insecticide. Bacillus thuringiensis (B.t.) is a facultative anaerobic, Gram-positive, motile, spore-forming bacterium. B.t. is accepted as a source of environment-friendly biopesticide. Farmers have applied B.t. as an insecticidal spray for control of lepidopteran and coleopteran pests for more than 30 years. The United States Environmental Protection Agency has considered B.t. sprays to be so safe that it has exempted them from the requirement of a tolerance (a standard for a maximum permissible residue limit on food).
There are other alternatives for delivery of B.t. toxin to target insects. B.t. toxin genes are inserted into microorganisms that are associated with the target insect habitat so that the transformed organisms will colonize and continue to produce enough quantities of toxin to prevent insect damage. Examples of these are the insertion of specific genes into bacteria that colonize plant leaf surface and roots externally, such as Pseudomonas cepacia, or internally, such as Clavibacter xyli. However, the release of living recombinant microorganisms causes many concerns and regulatory restrictions. Alternative methods of introducing genes into microorganisms have been developed to minimize potential horizontal gene flow to other bacterial species. These include using transposase-negative derivatives of Tn5 transposon, or suicide vectors that rely on homologous recombination for integration to be completed. There has also been a development of non-viable recombinant organisms that could increase toxin persistence in the field, such as products based on encapsulated B.t. toxins in P. fluorescens. This approach eliminates concerns associated with testing of living genetically engineered microorganisms.
B.t. proteins may be delivered in transgenic plants. Examples of such plants, called B.t. plants, protected from insect attack include cotton and corn. The U.S. Environmental Protection Agency has approved the commercial planting of B.t. cotton and corn since 1996.
The mechanism of action of the B.t. toxins proceeds through several steps that include solubilization of ingested crystal, proteolytic activation of the protoxins, binding of toxin to midgut receptors, and insertion of the toxin into the apical membrane to form ion channels or pores. Binding of the toxin to brush border membrane vesicles (BBMV) is supposed to be a two-step process involving reversible and irreversible steps. Multiple receptors may be involved in the process of toxin binding and membrane insertion.
Tabashnik et al. (Tabashnik 1992) described the phenomenon of synergy for B.t. Cry toxins and developed a formula for calculating synergy. Cry proteins are considered synergistic if the combined insecticidal potency is greater than the sum of the individual components. Cry1Aa and Cry1Ac are synergistic in bioassays against gypsy moth larvae (Lee and Dean, 1996). Other examples of B.t. synergy are reported for the Cry proteins of B.t. israelensis and combinations of spores and crystals against Plutella xylostella, the diamondback moth (Liu et al., 1998). Non-B.t. molecules are also known to synergize toxins. For example, ethylenediamine tetra acetic acid (EDTA) synergizes B.t. against P. xylostella. The synergy described herein is novel both in the nature of the synergistic molecule and the effect detected on important Lepidoptera larvae.
B.t. Toxin Receptors. Characterization of receptors from insect midgut and investigation of their interaction with Cry toxins provides an approach to elucidating toxin mode of action and designing improved Cry toxins for pest control. Most Cry toxin-binding midgut proteins identified to date belong to two main protein families: cadherin-like proteins and aminopeptidases. There is in vitro and in vivo evidence supporting the involvement of aminopeptidases in Cry1 toxicity against lepidopteran larvae. Aminopeptidases bind Cry toxins specifically allowing them to form pores in membranes (Masson et al., 1995; Sangadala et al., 2001; Sangadala et al., 1994). Recent studies provide evidence that aminopeptidase can function as receptors when expressed in cultured cells (Adang and Luo 2003) and insects (Gill and Ellar 2002; Rajagopal et al., 2002). Aminopeptidases do not always confer susceptibility to Cry toxins when expressed in heterologous systems (Banks et al., 2003; Simpson and Newcomb 2000).
Cadherin-like proteins are a class of Cry1 receptor proteins in lepidopteran larvae. Bombyx mori, the silkmoth, has a 175-kDa cadherin-like protein called BtR175 that functions as a receptor for Cry1Aa and Cry1Ac toxins on midgut epithelial cells (Hara et al., 2003; Nagamatsu et al., 1999). M. sexta has a 210-kDa cadherin-like protein, called Bt-R1, that serves as a receptor for Cry1A toxins (Bulla 2002a, b; Vadlamudi et al., 1993; Vadlamudi et al., 1995). Bt-R1, binds Cry1Aa, Cry1Ab, and Cry1Ac toxins on ligand blots (Francis and Bulla 1997). Purified membranes from COS cells expressing Bt-R1 bound all three Cry1A toxins in binding assays and ligand blots (Keeton and Bulla 1997). Furthermore, expression of Bt-R1 on the surface of COS7 cells led to toxin-induced cell toxicity as monitored by immunofluorescence microscopy with fixed cells (Dorsch et al., 2002).
Cadherin-like Bt-R1 protein has been suggested to induce a conformational change in Cry1Ab that allows the formation of a pre-pore toxin oligomer and increases binding affinity for aminopeptidase (Bravo et al. 2004). In Bombyx mori, the cadherin-like protein BtR175 serves as a Cry1Aa receptor (Nagamatsu et al., 1998). Sf9 cells expressing BtR175 swell after exposure to Cry1Aa toxin, presumably due to formation of ion channels in cell membranes (Nagamatsu et al. 1999). When expressed in mammalian COS7 cells, BtR175 induced susceptibility to Cry1Aa (Tsuda et al., 2003).
Hua et al. (Hua et al. 2004) developed a fluorescence-based assay using Drosophila S2 cells to analyze the function of Manduca sexta cadherin (Bt-R1a) as a Cry1 toxin receptor. Bt-R1a cDNA that differs from Bt-R1 by 37 nucleotides and two amino acids and expressed it transiently in Drosophila melanogaster, Schneider 2 (S2) cells (Hua et al. 2004). Cells expressing Bt-R1a bound Cry1Aa, Cry1Ab, and Cry1Ac toxins on ligand blots, and in saturation binding assays. More Cry1Ab was bound relative to Cry1Aa and Cry1Ac, though each Cry1A toxin bound with high-affinity (Kd values from 1.7 nM to 3.3 nM). Using fluorescent microscopy and flow cytometry assays, (Hua et al. 2004) showed that Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1Ba, killed S2 cells expressing Bt-R1a cadherin. These results demonstrated that M. sexta cadherin Bt-R1a functions as a receptor for the Cry1A toxins in vivo and validates our cytotoxicity assay for future receptor studies.
Involvement of a cadherin-superfamily gene disruption in resistance to Cry1Ac has been described for a laboratory resistant strain of Heliothis virescens (Gahan et al., 2001). The encoded protein, called HevCaLP, has the binding properties expected for a Cry1A receptor (Jurat-Fuentes et al. 2004). Similarly, Pectinophora gossypiella larvae with resistance alleles in genes encoding a cadherin-like protein were resistant to Cry1A toxins (Morin et al., 2003).
B.t. toxins bind to specific regions on cadherin-like proteins. Regions of domain II of Cry1A toxins are involved in binding to Bt-R1 (Gomez et al., 2002; Gomez et al., 2001). The first toxin binding region identified in Bt-R1 was a stretch of seven amino acid residues located in the cadherin repeat seven (CR7) (Gomez et al. 2002; Gomez et al. 2001). (Dorsch et al. 2002) identified a second Cry1Ab binding region within cadherin repeat 11 (CR11) in Bt-R1. Recombinant and synthetic peptides containing both amino acid sequences inhibited Cry1Ab toxicity in vivo when fed to M. sexta larvae (Dorsch et al. 2002; Gomez et al. 2001), demonstrating their involvement in toxin action. Previously, two Bt-R1 toxin-binding regions in CR 7 (Gomez et al. 2001) and 11 (Dorsch et al. 2002) were proposed as functional receptor sites. U.S. Ser. No. 60/538,753 entitled “Novel Binding Domain of Cadherin-like Toxin Receptor,” by Adang et al., under Attorney Docket No. UGR-104P, identifies an additional binding site recognized by Cry toxins that functions as a receptor. This additional binding site, which is also a functional receptor region, is contained in the CR12-Membrane Proximal Extracellular Domain (MPED) of Bt-R1a (Hua et al. 2004). The HevCaLP protein of H. virescens has a Cry1Ac binding site at a comparable position (Xie et al. 2004), suggesting a conservation of binding sites between cadherins of different insect species.
There is no known report or suggestion of a B.t. toxin receptor or fragment thereof being fed, or otherwise administered, to an insect pest, with or without a B.t. protein, in order to kill or otherwise prevent the insect from feeding on a plant. Previous competitive-binding studies suggest that there would be no change in toxicity (Gomez et al. 2002) or a reduction in toxicity due to competitive binding (Gomez et al. 2001; Dorsch et al. 2002; Gomez et al. 2003; Xie et al. 2004).